Fusion protein and its application in helicobacter pylori cag a antibody detection

Abstract

The present application relates to a fusion protein and its application in detection of Helicobacter pylori CagA antibody, and belongs to the technical field of biology.The present application provides a fusion protein, wherein the fusion protein comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO.1, or a derivative polypeptide with more than 80% homology with the amino acid sequence as shown in SEQ ID NO.1 and capable of being specifically combined with Helicobacter pylori CagA antibody; the fusion protein is capable of being specifically combined with Helicobacter pylori CagA antibody, and has a very high application prospect in detection of Helicobacter pylori CagA antibody.

Description

TECHNICAL FIELD

[0001] The present application relates to a fusion protein and its application in detection of Helicobacter pylori CagA antibody, and belongs to the technical field of biotechnology. BACKGROUND

[0002] Helicobacter pylori (Hp) is a gram-negative bacterium that is S-shaped or curved. In 1982, Barry J. Marshall and J. Robin Warren isolated and cultured Helicobacter pylori from human gastric mucosa specimens, thus successfully revealing its potential pathogenic mechanism, and they won the 2005 Physiology and Medicine Prize. In 1994, Helicobacter pylori was classified as a class I carcinogen by the World Health Organization. At present, the global infection rate of Helicobacter pylori is as high as about 50%, and the infection rate of Helicobacter pylori in developing countries is higher than that in developed countries.

[0003] Helicobacter pylori infection is a long-term and chronic process. After infection, the human body is generally difficult to clear spontaneously, resulting in lifelong infection, unless eradication treatment is performed, or the human gastric mucosa develops severe intestinal metaplasia, so that the bacterium is difficult to colonize, and Helicobacter pylori will automatically disappear in the human body. Studies have shown that long-term Helicobacter pylori infection can cause chronic gastritis, and as the infection degree deepens and the disease worsens, some patients will develop duodenal ulcers, and even gastric cancer. Therefore, it is crucial to detect whether the patient's blood contains Helicobacter pylori antibody in vitro to infer whether the patient has been infected or is currently infected with Helicobacter pylori, and then to intervene in the Helicobacter pylori infected population in a timely manner to protect the health of the patient. SUMMARY

[0004] To solve the above problems, the present application provides a fusion protein, which comprises:

[0005] (a) a polypeptide with an amino acid sequence as shown in SEQ ID NO. 1;

[0006] and / or (b) a derivative polypeptide with more than 80% homology with the amino acid sequence shown in SEQ ID NO. 1 and capable of specifically binding to Helicobacter pylori CagA antibody.

[0007] The present application also provides a nucleic acid molecule encoding the above-mentioned fusion protein.

[0008] In an embodiment of the present application, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO. 2.

[0009] The present application also provides a recombinant plasmid carrying the above-mentioned nucleic acid molecule.

[0010] In an embodiment of the present application, the vector of the recombinant plasmid is pET-28a vector, pMAL-C2 vector, pGEX-4T-1 vector, pET-32a vector or pET-Duet-1 vector.

[0011] The present application also provides a host cell, which carries the nucleic acid molecule described above.

[0012] Alternatively, the host cell carries the recombinant plasmid described above.

[0013] In an embodiment of the present application, the host cell is a prokaryotic cell or a eukaryotic cell.

[0014] In an embodiment of the present application, the host cell is Escherichia coli, yeast, lactic acid bacteria or Bacillus.

[0015] The present application also provides a method for preparing the fusion protein described above, which comprises inoculating the host cell described above into a culture medium for fermentation to obtain a fermentation broth, and then separating the fusion protein from the fermentation broth.

[0016] The present application also provides a Helicobacter pylori CagA antibody detection kit, which uses the fusion protein described above as a detection antigen of Helicobacter pylori CagA antibody.

[0017] The present application also provides a method for detecting Helicobacter pylori CagA antibody, which uses the Helicobacter pylori CagA antibody detection kit described above to detect a sample to be tested.

[0018] In an embodiment of the present application, the sample to be tested is whole blood or serum.

[0019] The present application also provides the use of the fusion protein described above or the kit described above or the method described above in the detection of Helicobacter pylori CagA antibody.

[0020] The present application also provides a Helicobacter pylori CagA antibody detection test paper, which uses the fusion protein described above as a detection antigen of Helicobacter pylori CagA antibody.

[0021] In an embodiment of the present application, the test paper comprises a chromatography membrane; one end of the chromatography membrane is connected to an adsorption pad; the adsorption pad carries the fusion protein with an immune label and the first antibody with an immune label; the chromatography membrane is provided with a detection line and a quality control line in sequence along the liquid chromatography direction; the fusion protein is coated on the detection line; the second antibody is coated on the quality control line; and the second antibody can specifically bind to the first antibody.

[0022] In an embodiment of the present application, the loading amount of the fusion protein on the colloidal gold adsorption pad is 0.1-1 μg / cm2.2 .

[0023] In one embodiment of the present application, the loading amount of the fusion protein on the colloidal gold adsorption pad is 0.5 μg / cm 2 .

[0024] In one embodiment of the present application, the loading amount of the first antibody on the colloidal gold adsorption pad is 0.1-5 μg / cm 2 .

[0025] In one embodiment of the present application, the loading amount of the first antibody on the colloidal gold adsorption pad is 0.3 μg / cm 2 .

[0026] In one embodiment of the present application, the detection line is coated at the detection line position by the fusion protein solution with a concentration of 0.5-1.5 mg / mL and a liquid amount of 0.5-1.5 μL / cm.

[0027] In one embodiment of the present application, the detection line is coated at the detection line position by the fusion protein solution with a concentration of 1 mg / mL and a liquid amount of 1 μL / cm.

[0028] In one embodiment of the present application, the quality control line is coated at the quality control line position by the second antibody solution with a concentration of 1-2 mg / mL and a liquid amount of 1-2 μL / cm.

[0029] In one embodiment of the present application, the quality control line is coated at the quality control line position by the second antibody solution with a concentration of 1.5 mg / mL and a liquid amount of 1.5 μL / cm.

[0030] In one embodiment of the present application, the immunolabeling is colloidal gold labeling, colloidal arsenic labeling, colloidal carbon labeling, colored latex labeling or fluorescent latex labeling.

[0031] In one embodiment of the present application, the first antibody is rabbit IgG antibody; and the second antibody is goat anti-rabbit IgG antibody.

[0032] In one embodiment of the present application, the test paper further comprises a base plate, a sample pad and a water absorption pad; and the base plate is provided with the sample pad, the adsorption pad, the chromatography membrane and the water absorption pad in sequence along the liquid chromatography direction.

[0033] The technical scheme of the present application has the following advantages:

[0034] The application provides a fusion protein, which comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO. 1, or a derived polypeptide with more than 80% homology with the amino acid sequence shown in SEQ ID NO. 1 and capable of being specifically combined with a Helicobacter pylori CagA antibody; the fusion protein can be specifically combined with the Helicobacter pylori CagA antibody and has a very high application prospect in Helicobacter pylori CagA antibody detection; the fusion protein has a high expression amount and a high uniform purity and is stable and not easy to be denatured. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 : Agarose gel electrophoresis result of amplification product.

[0036] Figure 2 : SDS-PAGE gel electrophoresis analysis result of purified protein.

[0037] Figure 3 : Overall structure schematic diagram of Helicobacter pylori CagA antibody detection test paper.

[0038] Figure 4 : Positive sample detection result.

[0039] Figure 5 : Negative sample detection result.

[0040] Figure 3 In the figure, the bottom plate 1, the sample pad 2, the adsorption pad 3, the chromatography membrane 4, the water absorption pad 5, the detection line T and the quality control line C. DETAILED DESCRIPTION

[0041] The following examples are provided to better further understand the application and are not limited to the best mode, do not constitute limitations on the content and protection scope of the application, and any person under the enlightenment of the application or the combination of the application with other prior art features obtains any product same or similar to the application, which falls within the protection scope of the application.

[0042] In the following examples, the specific experimental steps or conditions are not specified, and can be operated according to the conventional experimental steps described in the literature in the art or the conditions. The reagents or instruments used are not specified by the manufacturer, and are conventional reagent products that can be obtained by market purchase.

[0043] Example 1: Fusion protein and preparation thereof

[0044] The example provides a fusion protein capable of being specifically combined with a Helicobacter pylori CagA antibody; the amino acid sequence of the fusion protein is shown in SEQ ID NO. 1.

[0045] The preparation process of the fusion protein is as follows:

[0046] 1. Preparation of recombinant bacteria

[0047] A gene encoding the fusion protein HP2 with the nucleotide sequence shown in SEQ ID NO.2 was synthesized; the synthesized gene was amplified by PCR using upstream and downstream primers, and the restriction enzyme sites BamHI / XhoI were added to obtain the amplification product (the amplification product was recovered by agarose gel electrophoresis, and the agarose gel electrophoresis results of the amplification product are shown in the figure). Figure 1 The amplified product was ligated with the pMAl-C2 plasmid (purchased from Ubisoft) using restriction endonucleases BamHI / XhoI (purchased from NEB) to obtain the ligation product. The ligation product was transformed into Escherichia coli DH5α (purchased from Beijing Qingke Biotechnology Co., Ltd.) to obtain the transformation product. The transformation product was plated on LB solid medium (purchased from Solarbio) containing 100 μg / mL ampicillin and incubated upside down in a 37℃ incubator for 12 h to obtain transformants. Transformants were picked and inoculated into LB liquid medium (purchased from Solarbio) containing 100 μg / mL ampicillin and cultured in shake flasks at 37℃ and 180 rpm for 12 h. The plasmid was then extracted for restriction enzyme digestion verification and sequencing verification. If the verification was correct, the recombinant strain DH5α / pMAl-C2-hp2 was obtained.

[0048] 2. Fermentation of recombinant bacteria

[0049] Single colonies of the recombinant strain DH5α / pMAl-C2-hp2 obtained in step 1 were inoculated into 500 mL of LB liquid medium containing 100 μg / mL ampicillin and cultured with shaking at 37°C and 180 rpm for 12 h to obtain the culture medium. The entire culture medium was then transferred to 1000 mL of LB liquid medium containing 100 μg / mL ampicillin and cultured with shaking at 37°C and 220 rpm for 4 h until OD reached the target value. 600 When the concentration of α = 0.8, the temperature was lowered to 16℃ and cultured with shaking at 180 rpm for 1 h. After 1 h, IPTG (final concentration 1 mM) was added and fermented at 16℃ and 180 rpm for 12 h to obtain the fermentation broth.

[0050] 3. Purification and identification of fusion proteins

[0051] The fermentation broth was centrifuged at 8000 rpm for 10 min, and the bacterial cells were collected; 50 mL of PBS buffer (pH 7.4, concentration 0.01 M, purchased from Thermo Fisher) was added to the bacterial cells to resuspend them, obtaining a resuspension; the resuspension was broken by a high-pressure disrupter with a pressure of 800 bar, and the breaking was repeated three times, obtaining a broken liquid; the broken liquid was centrifuged at 12000 rpm for 40 min, and the supernatant was taken; the supernatant was purified by an MBP column (purchased from GE), obtaining a purified protein (the purified protein was subjected to SDS-PAGE gel electrophoresis analysis, and the analysis result is shown in Figure 2 );

[0052] The MBP column purification process is as follows:

[0053] (1) The storage solution in the MBP column was dropped out, 10 mL of water was used to wash the MBP column, and the operation was repeated twice;

[0054] (2) 10 mL of MBP equilibration solution was used to equilibrate the column, and the operation was repeated twice;

[0055] (3) The supernatant was added to the MBP column;

[0056] (4) 10 mL of equilibration solution was used to wash the column twice to remove impurities;

[0057] (5) 4 mL of elution solution was used to elute the protein, and the elution was collected.

[0058] The purified protein was subjected to secondary purification by using a Dextrin Sepharose High Performance (Lot: 10288766) kit (purchased from GE), obtaining a fusion protein, and the protein concentration detected by a UV spectrophotometer was 2.6 mg / mL.

[0059] Example 2: Colloidal gold test paper for detecting Helicobacter pylori CagA antibody and preparation thereof

[0060] As shown in Figure 3 , the present embodiment provides a Helicobacter pylori CagA antibody detection test paper, which comprises a base plate 1; the base plate 1 is provided with a sample pad 2, an adsorption pad 3, a chromatography membrane 4 and a water absorption pad 5 in sequence along a liquid chromatography direction; the adsorption pad 3 is loaded with a colloidal gold-labeled fusion protein and a colloidal gold-labeled rabbit IgG antibody according to the embodiment 1; the chromatography membrane 4 is provided with a test line T and a quality control line C in sequence along the liquid chromatography direction; the test line T is coated with the fusion protein according to the embodiment 1; and the quality control line C is coated with a goat anti-rabbit IgG antibody;

[0061] The loading amount of the colloidal gold-labeled fusion protein on the colloidal gold adsorption pad is 0.5 μg / cm 2 ; and the loading amount of the colloidal gold-labeled rabbit IgG antibody on the colloidal gold adsorption pad is 0.3 μg / cm2 ; the detection line is formed by coating the colloidal gold-labeled fusion protein solution with a concentration of 1 mg / mL at a liquid volume of 1 μL / cm at the detection line position; and the quality control line is formed by coating the colloidal gold-labeled goat anti-rabbit IgG antibody solution with a concentration of 1.5 mg / mL at a liquid volume of 1.5 μL / cm at the quality control line position.

[0062] The preparation method of the H. pylori CagA antibody test paper is as follows:

[0063] 1. Labeling

[0064] 1.1 Burning gold

[0065] 1) Pour 792 mL of process water into a triangular flask, and use a pipette to transfer 8 mL of a 2% (w / v, g / 100 mL) chlorauric acid (purchased from Aldrich) aqueous solution into the triangular flask, so that the final concentration of chlorauric acid is 0.02% (w / v), shake well, and place on an electric stove with a power of 2000 watts to heat to complete boiling;

[0066] 2) After complete boiling, quickly add 15 mL of 1% (w / v) trisodium citrate (purchased from Aldrich) solution to the triangular flask; continue heating, at which time it can be observed that the light yellow chlorauric acid aqueous solution quickly turns gray after the addition of trisodium citrate, and then turns black, and then gradually stabilizes into red, the whole process takes 2-3 minutes, and the deep red color does not change after 10 minutes, and heating is stopped;

[0067] 3) Cool the colloidal gold to room temperature (25°C), and dilute to the initial volume with process water, and reserve for use, the bottle of colloidal gold is clear and transparent, and a double gold solution is obtained.

[0068] 1.2 Adjusting pH

[0069] 1) Take a 10 mL test tube, and use a syringe to take 1 mL of the double gold solution prepared in step 1.1 into the test tube;

[0070] 2) Take 1 μL of 0.2 mol / L K2CO3 and 1 μL of antigen (the antigen is the purified fusion protein of Example 1 and rabbit IgG with a stock concentration of 16.8 mg / mL purchased from Sigma) and add them to the test tube, shake gently, and observe the color change, if the color changes, increase the amount of K2CO3 gradient until the color does not change (in this experiment, the pH of the fusion protein is set to 0.6%, and the pH of the rabbit IgG is set to 0.8%).

[0071] 1.3 Labeling of rabbit IgG (pH: 0.8%, final concentration 20 μg / mL)

[0072] 1) Take 60 mL of the twice gold solution prepared in step 1.1 and place it in a clean small beaker, stir it evenly with a magnetic stirrer, add 480 μL of 0.2 mol / L K2CO3, 71.5 μL of the rabbit IgG solution prepared in step 1.2, stir for 15 min, and add 300 μL of 10% (w / v) milk powder and stir for 15 min;

[0073] 2) Fill the solution prepared in step 1) in a clean centrifuge tube, weigh it on an electronic balance to make it even, and then centrifuge it at 12000 rpm for 15 min on a low-temperature high-speed centrifuge, take out the supernatant, discard it, collect the precipitate, and reconstitute the precipitate with 3 mL of 5% (v / v) TuIgG diluent (purchased from Sigma Company) to obtain the labeled rabbit IgG solution.

[0074] 1.4 Fusion protein labeling (pH: 0.6%, fusion protein final concentration 8 μg / mL)

[0075] 1) Take 50 mL of the twice gold solution prepared in step 1.1 and place it in a clean small beaker, stir it evenly with a magnetic stirrer, add 300 μL of 0.2 mol / L K2CO3, (50 mL*8 μg / mL) / 2.6 mg / mL of the fusion protein solution prepared in step 1.2 (153.8 μL), stir for 15 min, and add 250 μL of 10% (w / v) milk powder and stir for 15 min;

[0076] 2) Fill and balance in a clean centrifuge tube, then centrifuge at 12000 rpm for 15 min on a low-temperature high-speed centrifuge, take out the supernatant, discard it, collect the precipitate, reconstitute the precipitate to 2 mL with 5% (v / v) TuIgG diluent (purchased from Sigma Company), then add 400 μL of the labeled rabbit IgG solution, and dilute to 3 mL with the reconstitution solution (0.01 M PBS buffer solution with 2% (w / v) BSA and 0.02% (w / v) thiomersal added), to obtain the labeled fusion protein solution;

[0077] 3) Take one colloidal gold adsorption pad, spray the gold line at a concentration of 3.0 μL / cm, dry it for 4 h in the interval, and store it.

[0078] 2, Coating

[0079] 2.1 T line

[0080] Example: Prepare 100 μL of T line, coating concentration 0.8 mg / mL, fusion protein concentration 2.6 mg / mL.

[0081] Amount of fusion protein: (100 μL*0.8 mg / mL) / 2.6 mg / mL of the fusion protein solution prepared in step 1.2, 30.8 μL;

[0082] Alcohol: 100 μL / 10, 10 μL;

[0083] Base solution (0.01M PBS buffer + 2% (w / v) trehalose): 100 μL - amount of fusion protein - amount of alcohol, 59.2 μL.

[0084] The above solution was sampled into 500 μL EP tubes in turn, and vortexed and mixed.

[0085] 2.2C line

[0086] Example: 100 μL of C line, envelope concentration 0.8 mg / mL, goat anti-rabbit IgG (purchased from Sigma Company) concentration 12.8 mg / mL.

[0087] Amount of antigen: (100 μL * 1.5 mg / mL) / 12.8 mg / mL, 11.7 μL;

[0088] Alcohol: 100 μL / 10, 10 μL;

[0089] Base solution (0.01M PBS buffer + 2% (w / v) trehalose): 100 μL - amount of fusion protein - amount of alcohol, 59.2 μL.

[0090] The above solution was sampled into 500 μL EP tubes in turn, and vortexed and mixed.

[0091] 2.3 Treatment

[0092] The prepared C, T line solution was coated on the coating machine, and after coating, it was placed in a refrigerator at 4°C for not less than 4 h, soaked with five treatment liquids for 30 min, pasted with film, and dried under dry conditions.

[0093] 3, Compound

[0094] A handle paper was pasted on one side of the film C line, with a distance of 1 mm from the film surface, and a gold strip was pasted on one side of the film T line, and an HP carrier was pasted, and an instant sticker was pasted on the gold, and a test paper strip with a width of 3.0 mm was cut, to obtain a Helicobacter pylori CagA antibody test paper.

[0095] Example 3: Application of Helicobacter pylori CagA antibody detection colloidal gold test paper

[0096] 1000 negative samples and 100 positive samples (blood samples, from Henan Center for Disease Control and Prevention) were taken, and the Helicobacter pylori CagA antibody test paper of Example 2 was used to detect Helicobacter pylori CagA antibody in the samples. If one color band appeared on each of the detection line and the quality control line, it was positive, and if only one color band appeared on the quality control line, it was negative. The detection results are as follows:

[0097] The negative compliance rate is greater than 95% ( Figure 4 (This refers to the test results of partially negative samples); the positive detection rate is greater than 96% ( Figure 5 (Results of partial positive samples). It is evident that the accuracy of using the Helicobacter pylori CagA antibody test strip of Example 2 for detecting Helicobacter pylori CagA antibodies in samples is high. The Helicobacter pylori CagA antibody test strip of Example 2, by detecting the presence of Helicobacter pylori antibodies in patient blood samples in vitro, is highly promising for inferring whether a patient has been infected or is currently infected with Helicobacter pylori.

[0098] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention. sequence list <110> Nantong Yishi Biotechnology Co., Ltd. <120> Fusion Protein and Its Application in the Detection of Helicobacter pylori CagA Antibody <160> 4 <170> PatentIn version 3.3 <210> 1 <211> 676 <212> PRT <213> Artificial sequence <400> 1 Pro Gln Gln Phe Ile Asn Asn Leu Gln Val Ala Phe Leu Lys Val Asp 1 5 10 15 Asn Ala Val Ala Ser Tyr Asp Pro Asp Gln Lys Pro Ile Val Asp Lys 20 25 30 Asn Asp Arg Asp Asn Arg Gln Ala Phe Glu Gly Ile Ser Gln Leu Arg 35 40 45 Glu Glu Tyr Ser Asn Lys Ala Ile Lys Asn Pro Thr Lys Lys Asn Gln 50 55 60 Tyr Phe Ser Asp Phe Ile Glu Lys Ser Asn Asp Leu Ile Asn Lys Asp 65 70 75 80 Asn Leu Ile Asp Val Glu Ser Ser Thr Glu Ser Phe Arg Lys Phe Gly 85 90 95 Asp Gln Arg Tyr Arg Ile Phe Thr Ser Trp Val Ser His Gln Asn Asp 100 105 110 Pro Ser Lys Ile Asn Thr Arg Ser Ile Arg Asn Phe Met Glu His Thr 115 120 125 Ile Gln Pro Pro Ile Pro Asp Asp Lys Glu Lys Ala Glu Phe Leu Lys 130 135 140 Ser Ala Lys Gln Ser Phe Ala Gly Ile Ile Ile Gly Asn Gln Ile Arg 145 150 155 160 Thr Asp Gln Lys Phe Met Gly Val Phe Asp Glu Ser Leu Lys Glu Arg 165 170 175 Gln Glu Ala Glu Lys Asn Gly Gly Pro Thr Gly Gly Asp Trp Leu Asp 180 185 190 Ile Phe Leu Ser Phe Ile Phe Asp Lys Lys Gln Ser Ser Asp Val Lys 195 200 205 Glu Ala Ile Asn Gln Glu Pro Val Pro His Val Gln Pro Asp Ile Ala 210 215 220 Thr Ser Thr Thr His lie Gin Gly Leu Pro Pro Glu Ser Arg Asp Leu 225 230 235 240 Leu Asp Glu Arg Gly Asn Phe Ser Lys Phe Thr Leu Gly Asp Met Glu 245 250 255 Met Leu Asp Val Glu Gly Val Ala Asp Met Asp Pro Asn Tyr Lys Phe 260 265 270 Asn Gin Leu Leu lie His Asn Asn Ala Leu Ser Ser Val Leu Met Gly 275 280 285 Ser His Asp Gly lie Glu Pro Glu Lys Val Ser Leu Leu Tyr Ala Gly 290 295 300 Asn Gly Gly Phe Gly Asp Lys His Asp Trp Asn Ala Thr Val Gly Tyr 305 310 315 320 Lys Asp Gin Gin Gly Asn Asn Val Ala Thr lie lie Asn Val His Met 325 330 335 Lys Asn Gly Ser Gly Leu Val lie Ala Gly Gly Glu Lys Gly lie Asn 340 345 350 Asn Pro Ser Phe Tyr Leu Tyr Lys Glu Asp Gin Leu Thr Gly Ser Gin 355 360 365 Arg Ala Leu Ser Gin Glu Glu lie Leu Asn Lys lie Asp Phe Met Glu 370 375 380 Phe Leu Ala Gin Asn Asn Ala Lys Leu Asp Asn Leu Ser Glu Lys Glu 385 390 395 400 Lys Glu Lys Phe Arg Asn Glu He Lys Asp Phe Gin Lys Asp Ser Lys 405 410 415 Pro Tyr Leu Asp Ala Leu Gly Asn Asp Arg He Thr Phe Val Ser Lys 420 425 430 Lys Asp Pro Lys His Ser Ala Leu He Thr Glu Phe Asn Lys Gly Asp 435 440 445 Leu Ser Tyr Thr Leu Lys Asp Tyr Gly Lys Lys Ala Asp Lys Ala Leu 450 455 460 Asp Arg Glu Lys Asn Val Thr Leu Gin Gly Ser Leu Lys His Asp Gly 465 470 475 480 Val Met Phe Val Asn Tyr Ser Asn Phe Lys Tyr Thr Asn Ala Ser Lys 485 490 495 Ser Pro Asn Lys Gly Val Gly Val Thr Asn Gly Val Ser His Leu Glu 500 505 510 Ala Gly Phe Ser Lys Val Ala Val Phe Asn Leu Pro Asn Leu Asn Asn 515 520 525 Leu Ala lie Thr Ser Val Val Arg Arg Asp Leu Glu Asp Lys Leu lie 530 535 540 Ala Lys Gly Leu Pro Pro Gin Glu Ala Asn Lys Leu Val Lys Gly Phe 545 550 555 560 Leu Ser Ser Asn Lys Glu Leu Val Gly Lys Ala Leu Asn Phe Asn Lys 565 570 575 Ala Val Ala Glu Ala Lys Asn Thr Gly Asn Tyr Asp Glu Val Lys Arg 580 585 590 Ala Gin Lys Asp Leu Glu Lys Ser Leu Lys Lys Arg Glu Arg Leu Glu 595 600 605 Lys Asp Val Ala Lys Asn Leu Glu Ser Lys Ser Gly Asn Lys Asn Lys 610 615 620 Met Glu Ala Lys Ser Gin Ala Asn Ser Gin Lys Asp Glu lie Phe Ala 625 630 635 640 Leu lie Asn Lys Glu Ala Asn Arg Asp Ala Arg Ala lie Ala Tyr Thr 645 650 655 Gln Asn Leu Lys Gly lie Lys Arg Glu Leu Ser Asp Lys Leu Glu Asn 660 665 670 Ile Asn Lys Asp 675 <210> 2 <211> 2043 <212> DNA <213> Artificial Sequence <400> 2 ccgcagcagt tcatcaacaa cctccaagtt gcgttcctga aagttgacaa cgctgttgct 60 tcttacgacc cggaccagaa accgatagtt gacaaaaacg accgtgacaa tcgtcaggcg 120 ttcgaaggta tctcgcagct gcgtgaagaa tactctaaca aagctatcaa aaacccgacc 180 aaaaaaaacc agtacttctc tgacttcatc gaaaaatcta acgacctgat aaacaaagac 240 aacctgatag acgttgaatc ttctaccgaa tctttccgta aattcggtga ccagcgttac 300 cgtatcttca cctcttgggt ttctcaccag aacgacccgt ctaaaatcaa cacccgttct 360 atccgtaact tcatggaaca caccatccag ccgccgatac cggacgacaa agaaaaagct 420 gaatttctga aatctgctaa acagtctttc gctggtatca tcatcggtaa ccagatacgt 480 accgaccaga aattcatggg tgttttcgac gaatctctga aagaacgtca ggaagctgaa 540 aaaaacggtg gtccgaccgg tggtgactgg ctcgacatct tcctgagctt catcttcgac 600 aagaaacagt cttctgacgt taaagaagct atcaaccagg aaccggttcc gcacgttcag 660 ccggacatcg ctacctctac cacccacatc cagggtctgc cgccggaatc tcgtgacctg 720 ctggacgaac gtggtaactt ctctaaattc accctgggtg acatggaaat gctggacgtt 780 gaaggtgttg ctgacatgga cccgaactac aaattcaacc agctgctgat acacaacaac 840 gctctgtctt ctgttctgat gggttctcac gacggtatcg aaccggaaaa agtttctctg 900 ctgtacgctg gtaacggtgg tttcggtgac aaacacgact ggaacgctac cgttggttac 960 aaagaccagc agggtaacaa cgttgctacc atcatcaacg ttcacatgaa aaacggttct 1020 ggtctggtta tcgctggtgg tgaaaaaggt atcaacaacc cgtctttcta cctgtacaaa 1080 gaagaccagc tgaccggttc tcagcgtgct ctgtctcagg aagaaatcct gaacaaaatc 1140 gacttcatgg aatttctggc tcagaacaac gctaaactgg acaacctgtc tgaaaaagaa 1200 aaagaaaaat tccgtaacga aatcaaagac ttccagaaag actctaaacc gtacctggac 1260 gctctgggta acgaccgtat caccttcgtt tctaaaaaag acccgaaaca ctctgctctg 1320 ataaccgaat ttaacaaagg tgacctgtct tacaccctga aagactacgg taaaaaagct 1380 GACAAAGCTC TGGACCCTGA AAAACGTTAC CCTGCAGGTT CTCTGAAACA CGACGGT 1440 GTTATGTTCT GCTAATTACT CTAATTCAAA TACACCAACG CTTCTAAATC TCCGAACAAA 1500 GGTGTGGGCG TTACTAACGG TGTAAGCCAC CTGGAGGCAG GCTTCTCTAA AGTTGCGGTT 1560 TTCAACCTGC CGAACCCTGA ACAACCTGGC TATCACCTCT GTTTTGGTCG TGACCTGGAA 1620 GACAAACTGA TAGCTAAAGG TCTGCCGCCG CAGGAAGCTA ACAAAGTGTT AAAGTTTCT 1680 CTGTCTTCTA ACAAAGAACC TGGTTGGTAA AGCTCTGAAC TTCAACAAGC GTTGCTGAAC 1740 GCTAAAAACA CGGTAACGTA CGACGAAGTT AAACGTGCTC AGAAAGACCT GGAAAAATCT 1800 CTGAAAAAAC GTGAACGTCT GGAAAAAGAC GTTGCTAAAA CCTGGAATCT AAATCTGGTT 1860 ACAAAAACAA AATGGAAGCT AAATCTCAGG CTAACCTCAG AAAGACGAAT CTTGGCT 1920 CTGATAAACA AAGAAGCTAA CCCTGACGCT CGTGCTATCG CTTACACCCA GAACCTGAAA 1980 GCTTATCAAC GTGAACCGTC TGACAAACTG GAAAACATCA ACAAAGACCT GAAAGACTTC 2040 TCT 2043 <210> 3 <211> 36 <212> DNA <213> Artificial sequences <400> 3 cgcggatcca tgccgcagca gttcatcaac aacctc 36 <210> 4 <211> 36 <212> DNA <213> Artificial sequences <400> 4 ccgctcgagt taagagaagt ctttcaggtc tttgtt 36

Claims

1. A Helicobacter pylori CagA antibody detection kit, characterized in that, The kit uses the fusion protein as the detection antigen for Helicobacter pylori CagA antibody; the amino acid sequence of the fusion protein is shown in SEQ ID NO.

1.

2. A Helicobacter pylori CagA antibody detection strip, characterized in that, The test strip uses the fusion protein as the detection antigen for Helicobacter pylori CagA antibody; the amino acid sequence of the fusion protein is shown in SEQ ID NO.

1.

3. The antibody test strip as described in claim 2, characterized in that, The test strip includes a chromatography membrane; one end of the chromatography membrane is connected to an adsorption pad; the adsorption pad carries an immunolabeled fusion protein and an immunolabeled first antibody; a detection line and a control line are sequentially arranged on the chromatography membrane along the liquid chromatography direction; The detection line is coated with the aforementioned fusion protein; the quality control line is coated with a second antibody; the second antibody can specifically bind to the first antibody.

4. The antibody test strip as described in claim 3, characterized in that, The loading capacity of the fusion protein on the colloidal gold adsorption pad is 0.1–1 μg / cm³. 2 The loading capacity of the first antibody on the colloidal gold adsorption pad is 0.1–5 μg / cm³. 2 .

5. The antibody test strip as described in claim 4, characterized in that, The loading capacity of the fusion protein on the colloidal gold adsorption pad was 0.5 μg / cm³. 2 The loading capacity of the first antibody on the colloidal gold adsorption pad was 0.3 μg / cm³. 2 .

6. The antibody test strip as described in claim 3, characterized in that, The detection line is formed by coating the detection line position with a fusion protein solution of concentration of 0.5-1.5 mg / mL at a liquid volume of 0.5-1.5 μL / cm; the control line is formed by coating the control line position with a second antibody solution of concentration of 1-2 mg / mL at a liquid volume of 1-2 μL / cm.

7. The antibody test strip as described in claim 6, characterized in that, The detection line is formed by coating the detection line position with a fusion protein solution of 1 mg / mL at a liquid volume of 1 μL / cm; the control line is formed by coating the control line position with a second antibody solution of 1.5 mg / mL at a liquid volume of 1.5 μL / cm.

8. The antibody test strip as described in claim 3, characterized in that, The test strip also includes a base plate, a sample pad, and an absorbent pad; the base plate is provided with the sample pad, the adsorption pad, the chromatography membrane, and the absorbent pad in sequence along the liquid chromatography direction.

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